The overriding objective that will be pursued in this project is to develop new biocompatible fluorescent probes capable of providing facile detection of single-molecule events in living cells. In pursuit of this goal, under-explored and novel semiconductor nanocrystal quantum dot (NQD)-based probes will be synthesized, characterized with respect to their photophysical, structural and chemical properties, and screened to ascertain biocompatibility. NQDs offer high signal output, narrow bandwidth, improved stability with respect to photobleaching, broadband absorption for facile excitation, reasonably small size, and flexibility in surface chemistry for potentially achieving deliverability and physiological neutrality. The two NQD-based systems that will be developed here are (1) Near-infrared-emitting NQDs and (2) Lanthanide (Ln) doped NQDs, where the NQD serves as a sensitizer for Ln emission. We will target systems that provide emission from 600 - 1400 nm. This spectral region below 1000 nm is distinguished by a high transmittance through biological tissue and is worth extending farther into the infrared to 1400 nm for cellular studies, as interfering water absorption increases significantly only above this wavelength. Despite all the inherent advantages of NQD-based materials for optical imaging applications, several obstacles remain. Firstly, long-term single-NQD tracking in cells is hindered by fluorescence intermittency (blinking) that is characteristic of NQDs. Secondly, NQD biocompatibility is a concern for heavy-metal-containing NQDs or for NQDs that are improperly surface passivated. With respect to the first deficiency, though it has been postulated that the origin of blinking is related to charge transfer processes at the NQD surface, the experimental evidence is limited and the quantitative understanding of the connection between blinking and NQD charging is lacking. Without an experimentally validated understanding of this fundamental process, efforts to design and synthesize non-blinking NQDs are inherently impeded. We will perform steady-state and ultrafast spectroscopic studies to elucidate the mechanism of charging and correlate these results with single-NQD blinking studies. Results of spectroscopic studies will provide guidance for the design of photochemically stable structures that is anticipated to rely on inorganic heterostructuring (e.g., complex core/shell architectures). We will for the first time investigate blinking in infrared-emitting NQDs for which even rudimentary studies are lacking with the objective to understand the underlying mechanism and to develop synthetic strategies for its elimination. We will also investigate novel Ln-NQD coupled systems, in which the luminescence originates in the Ln dopant and is therefore not expected to exhibit blinking. The aim here will be to optimize the energy transfer process from the absorber NQD to the emitting Ln and, thereby, the signal output of the combined system. In parallel with these studies, we will address the second perceived deficiency of NQD-based fluorophores - insufficient biocompatibility - by investigating the toxicity and localization of our NQD-based probes in a variety of human cell lines. Similar to the blinking studies, the biocompatibility studies will provide valuable feedback in the design of probes possessing appropriate composition, surface passivation, and surface functionality. The ability to image real-time the location, activity and reactivity of biomolecules as they occur within living cells is fundamental to furthering biomedical science, including drug discovery, but currently available fluorescent molecular probes are not capable of providing for the routine study of molecules and molecular events. The advanced quantum dot based molecular probes that we propose to develop through a combination of fundamental physical, chemical and biological studies will enable the advances necessary for achieving the required optical molecular imaging capability. [unreadable] [unreadable] [unreadable]